DESCRIPTION:This is the first renewal application from a beginning investigator, Dr. Elizabeth Goodwin of Northwestern University. Dr. Goodwin is seeking five years of support to continue her studies of the translational regulation of the tra-2 mRNA in the nematode C. elegans. tra-2 is a member of a cascade of genes that controls C. elegans' sexual identity. The presence of the tra-2 gene product, a large transmembrane protein, promotes female cell fates (in XX animals). Translation and poly(A) length of the tra-2 mRNA are regulated by two 28 nt elements, DREs, located in the mRNA's 3'-UTR. These elements are specifically bound by a factor designated DRF which is thought to repress translation and limit poly(A) length. Three genes, laf-1, tra-3, and tra-1 regulate DRE control of tra-2 expression. laf-1 is required for translational repression, and has thus been a candidate for DRF. tra-3 and tra-1 act to free tra-2 from DRE regulation. Interestingly, tra-1 also appears to be a transcriptional regulator in the sex determination pathway. Yet another gene, glp-1, regulates tra-2 translation by a DRE-independent pathway. The proposed experiments will attempt to elucidate the mechanism by which tra-2 translation is governed by these factors and its 3'-UTR. Seven specific aims will address three general questions: 1) What are the precise cis-acting elements and how do they regulate translation? This question will be addressed by using reporter assays, mutational analysis, and RNA gel shift assays to identify the nucleotides required for DRE and glp-1 regulation, to determine whether glp-1 regulation also affects poly(A) length, and to test whether tra-1 expression is also regulated by 3' DREs or other UTR elements. 2) What gene products regulate translation and how? Here, the PI will utilize molecular (e.g., three-hybrid) and biochemical techniques to identify, clone, and characterize DRF and laf-1. Subsequently, genetics, reporter constructs, and gel shifts will be employed to characterize the mechanisms by which tra-1, tra-3, and glp-1 regulate tra-2 translation and to identify the genes that affect tra-1 translational control. 3) How do 3'-UTR controls regulate translation in a manner that allows correct temporal and spatial regulation of developmental fate? Here, the PI will use genetics, reporter constructs, and gel shift analyses to determine how laf-1, tra-3, tra-1, and glp-1 control the pattern of tra-2 expression during development.